Single-Base Resolution: Increasing the Specificity of the CRISPR-Cas System in Gene Editing

The CRISPR-Cas system holds a great promise in the treatment of diseases caused by genetic variations. The Cas protein, an RNA-guided programmable nuclease, generates a double-strand break at precise genomic loci. However, employing the CRISPR-Cas system to distinguish between single-nucleotide variations is challenging. The promiscuity of the guide-RNA (gRNA) and its mismatch tolerance make allele-specific targeting an elusive goal. This review presents a meta-analysis of previous studies reporting position-dependent mismatch tolerance within the gRNA. We also examine the conservativity of the seed sequence, a region within the gRNA with stringent sequence dependency, and propose the existence of a sub-region within the seed sequence with a higher degree of specificity. In addition, we summarize the reports on high-fidelity Cas nucleases with improved specificity, and compare the standard gRNA design methodology to the SNP-derived PAM approach, an alternative method for allele-specific targeting. Combining the two methods may be advantageous in designing CRISPR based therapeutics and diagnostics for heterozygous patients.